The goal of this research is to pinpoint substances that exert physiological control of hepatocyte proliferation and to define their operant mechanisms. On the basis of our prior and current studies, we postulate a regulatory signal complex comprised of certain hormone/growth factor/nutrient combinations interacting synergistically. Freshly isolated adult rat hepatocytes in a serum-free, artificial medium are stimulated to synthesize DNA (3H-thymidine incorporation) by epidermal growth factor (EGF) + insulin. This affords a "standard stimulus" for comparison with other possible physiological liver growth regulatory substances, either stimulatory or inhibitory. We are employing three general approaches: (1)\we have found that vasopressin (AVP) can amplify the stimulus provided by low concentrations of insulin plus EGF or normal rat serum; the degree of responsiveness appears to be influenced by changes in the metabolic state of the cells. In this respect AVP may resemble glucagon, which is stimulatory in the presence of some metabolic substrates and inhibitory with others. Hepatic effects of AVP acting through the V-1 type receptor are probably mediated by Ca2+. Studies with AVP analogues should be informative. Evidence fromfibroblasts points to an important influence of AVP on Na+ flux in relation to growth stimulation. (2)\Normal rat serum stimulates hepatocyte proliferation about as effectively as insulin + EGF, and half of this activity is derived from a heat-labile component of the platelets that is not "platelet-derived growth factor" (PDGF). Hepatectomized rat serum does not differ from normalserum in our system, despite much in vivo evidence that liver regeneration is controlled by humoral factors. Others using a less enriched medium do find a difference. We are attempting to discover why we find none, why normal rat serum is so stimulatory, and whether the serum is merely compensating for deficiencies in the medium or substratum. Attempts to define and characterize the active components of rat serum are continuing. (3)\We are also continuing to explore effects of other nutrients, hormones, and growth factors, as this approach has proved fruitful especially in uncovering the enormous potentiating effect of pyruvate when added in conjunction with other growth-promoting substances. All of this work will largely follow guidelines set forth in the original work. (J)